Project Summary/Abstract: The highly conserved COMPASS (COMplex of Proteins ASsociated with Set1) family of methyltransferases is responsible for the implementation of histone H3 lysine 4 (H3K4) methylation, an epigenetic mark associated with transcriptionally active chromatin. Multiple genome-wide sequencing efforts reported that subunits of the COMPASS family are frequently mutated in large number of cancers, neurological disorders, and other diseases. Therefore, understanding COMPASS function will lend important insights into the underlying mechanisms of disease pathogenesis, which is necessary for the development of effective targeted therapeutics. The H3K4 methylase Set1A is one of six COMPASS family members identified in mammals, and has been shown to be involved in bulk H3K4 di- and trimethylation (H3K4me2/me3 respectively) across the genome. Previous studies demonstrated that the loss of Set1A protein resulted in embryonic lethality and embryonic stem cell (ESC) proliferation defects, suggesting that Set1A is required for this process; however, the molecular context in which Set1A functions is primarily unknown. To distinguish between a role for full-length Set1A vs. its catalytic domain, the catalytic domain of Set1A was deleted in ESCs, which resulted in the very surprising discovery that its enzymatic function appears to be dispensable for ESC viability and self-renewal. However, ESCs bearing catalytically dead Set1A seemed unable to properly differentiate, suggesting multiple roles for Set1A and its catalytic activity in ESC self-renewal and subsequent differentiation. Together, these preliminary data support further investigation into the role of Set1A in regulating stem cell pluripotency and development. Based on these results, the first aim of this proposal will further examine the enzymatic function of Set1A by analyzing transcriptional and H3K4 methylation changes in Set1A catalytic mutants during ESC differentiation and mouse development. The second aim will determine the critical domains of Set1A protein in maintaining ESC self-renewal via a conditional knockout and overexpression system. The third aim will further explore the protein interaction network of Set1A in ESCs to reveal novel mechanisms underlying the functions of Set1A in early development. This work will illuminate the basic functional significance of Set1A in stem cells and in development, which will elucidate its disease liability and ultimately facilitate treatment development.